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Oxalis features: independent control of pO2, pH and the liquid flowrate. pO2 equilibration time in the medium: 3 minutes. pO2 accuracy: 3 mmHg. Flowrate as low as 1 μL min−1 to avoid shear stress.

Muscle-on-chip devices aim to recapitulate the physiological characteristics of in vivo muscle tissue and so maintaining levels of oxygen transported to cells is essential for cell survival and for providing the normoxic conditions experienced in vivo . We use finite-element method numerical modelling to describe oxygen transport and reaction in a proposed three-dimensional muscle-on-chip bioreactor with embedded channels for muscle cells and growth medium. We determine the feasibility of ensuring adequate oxygen for muscle cell survival in a device sealed from external oxygen sources and perfused via medium channels. We investigate the effects of varying elements of the bioreactor design on oxygen transport to optimize muscle tissue yield and maintain normoxic conditions. Successful co-culturing of muscle cells with motor neurons can boost muscle tissue function and so we estimate the maximum density of seeded neurons supported by oxygen concentrations within the bioreactor. We show that an enclosed bioreactor can provide sufficient oxygen for muscle cell survival and growth. We define a more efficient arrangement of muscle and perfusion chambers that can sustain a predicted 50% increase in maximum muscle volume per perfusion vessel. A study of simulated bioreactors provides functions for predicting bioreactor designs with normoxic conditions for any size of perfusion vessel, muscle chamber and distance between chambers.

Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy and optical tweezer-based approaches, we demonstrate that TNTs forming through the outward extension of actin (not through cellular dislodgement) achieve distances greater than the mean length of filopodia, and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, limiting TNT occurrence. Proteomic analysis using Epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form TNTs, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour TNT formation. Thus, this work reinforces a general principle for actin network control for cellular protrusions where simple shifts in the balance between processes that inhibit actin growth versus those that promote growth dictate protrusion formation and the ultimate length scales protrusions achieve.

Organ-on-chip and tumor-on-chip microfluidic cell cultures represent a fast-growing research field for modelling organ functions and diseases, for drug development, and for promising applications in personalized medicine. Still, one of the bottlenecks of this technology is the analysis of the huge amount of bio-images acquired in these dynamic 3D microenvironments, a task that we propose to achieve by exploiting the interdisciplinary contributions of computer science and electronic engineering. In this work, we apply this strategy to the study of oncolytic vaccinia virus (OVV), an emerging agent in cancer immunotherapy. Infection and killing of cancer cells by OVV were recapitulated and directly imaged in tumor-on-chip. By developing and applying appropriate image analysis strategies and advanced automatic algorithms, we uncovered synergistic cooperation of OVV and immune cells to kill cancer cells. Moreover, we observed that the kinetics of immune cells were modified in presence of OVV and that these immune modulations varied during the course of infection. A correlation between cancer cell infection and cancer-immune interaction time was pointed out, strongly supporting a cause-effect relationship between infection of cancer cells and their recognition by the immune cells. These results shed new light on the mode of action of OVV, and suggest new clinical avenues for immunotherapy developments.

Microfluidics has provided clinicians with new technologies to detect and analyze circulating tumor biomarkers in order to further improve their understanding of disease mechanism, as well as to improve patient management. Among these different biomarkers, circulating tumor cells have proven to be of high interest for different types of cancer and in particular for breast cancer. Here we focus our attention on a breast cancer subtype referred as HER2-positive breast cancer, this cancer being associated with an amplification of HER2 protein at the plasma membrane of cancer cells. Combined with therapies targeting the HER2 protein, HER2-HER3 dimerization blockade further improves a patient’s outcome. In this work, we propose a new approach to CTC characterization by on-chip integrating proximity ligation assay, so that we can quantify the HER2-HER3 dimerization event at the level of single CTC. To achieve this, we developed a microfluidic approach combining both CTC capture, identification and HER2-HER3 status quantification by Proximity Ligation Assay (PLA). We first optimized and demonstrated the potential of the on-chip quantification of HER2-HER3 dimerization using cancer cell lines with various levels of HER2 overexpression and validated its clinical potential with a patient’s sample treated or not with HER2-targeted therapy.

Correction for ‘Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions’ by Marine Verhulsel et al., Lab Chip, 2021, 21, 365–377, https://doi.org/10.1039/d0lc00672f.

During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule is a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.

The progress of food in the gastrointestinal (GI) tract is driven by a peristaltic motion generated by the muscle belt surrounding the GI tract. In turn, the response of the intestinal epithelial cells to the peristaltic stresses affects the dynamics of the epithelial structure. In this work, we study the effect of cyclic stretching (0.125 Hz, 10% strain) on the spatial organization of the intestinal epithelium using intestinal cells deposited on a flat elastomeric substrate to mimic the peristaltic motion in vitro. A confluent monolayer of Caco-2 cells is grown on a PDMS chip to probe the morphological and orientational response of the tissue to cyclic stretching. The PDMS chips are either covalently or non-covalently coated with laminin to recapitulate the basement membrane. We observe a significant orientational response where the cells rearrange their long axes perpendicular to the stretching direction for both coating conditions. The experiment is modeled by a vertex model where the cells store elastic energy with varying strain and effectively have a rotational diffusive motion through rearrangements of their shapes. The model also predicts a transition between the perpendicular orientation and orientation at an oblique angle determined by the level of the cell elastic anisotropy. It provides a general framework to study cell response and relaxation dynamics under cyclic stretching across different cell types. We also discuss potential relevance of peristalsis in determining planar cell polarity in 3D architectures.

International audience; Miniaturized devices for the extraction of DNA have been used for assessing genetic material in biological, forensic and environmental samples. However, the ability to isolate trace amounts of highly fragmented DNA from biological fluids remains a challenge. The current work reports a microfluidic approach that combines on line a dynamic magnetic extraction procedure with droplet-based digital PCR (ddPCR). This strategy maximizes the surface area for DNA binding within the chip, in order to capture short DNA fragments, with the possibility of recovering the purified samples into picoliter volumes for high sensitivity mutation detection. The application of this technology to capture circulating cell-free DNA (ccfDNA) from serum samples of cancer patients is demonstrated herein, with efficiencies comparable to standard column-based DNA extraction methods. This technology uses lesser amounts of required material and reagents, and has a higher potential for automation and multiplex DNA analysis. Furthermore, this approach can also be extended for the detection of other circulating biomarkers, such as nucleic acid sequences with aberrant methylation patterns or miRNA.

International audience; The ability to specifically block or degrade cytosolic targets using therapeutic proteins would bring tremendous therapeutic opportunities in cancer therapy. Over the last few years, significant progress has been made with respect to tissue targeting, cytosolic delivery, and catalytic inactivation of targets, placing this aim within reach. Here, we developed a mathematical model specifically built for the evaluation of approaches towards cytosolic protein delivery, involving all steps from systemic administration to translocation into the cytosol and target engagement. Focusing on solid cancer tissues, we utilized the model to investigate the effects of microvascular permeability, receptor affinity, the cellular density of targeted receptors, as well as the mode of activity (blocking/degradation) on therapeutic potential. Our analyses provide guidance for the rational optimization of protein design for enhanced activity and highlight the importance of tuning the receptor affinity as a function of receptor density as well as the receptor internalization rate. Furthermore, we provide quantitative insights into how enzymatic cargoes can enhance the distribution, extent, and duration of therapeutic activity, already at very low catalytic rates. Our results illustrate that with current protein engineering approaches, the goal of delivery of cytosolic delivery of proteins for therapeutic effects is well within reach.

International audience; Bone is the most frequent metastasis site for breast cancer. As well as dramatically increasing disease burden, bone metastases are also an indicator of poor prognosis. One of the main challenges in investigating bone metastasis in breast cancer is engineering in vitro models that replicate the features of in vivo bone environments. Such in vitro models ideally enable the biology of the metastatic cells to mimic their in vivo behavior as closely as possible. Here, taking benefit of cutting-edge technologies both in microfabrication and cancer cell biology, we have developed an in vitro breast cancer bone-metastasis model. To do so we first 3D printed a bone scaffold that reproduces the trabecular architecture and that can be conditioned with osteoblast-like cells, a collagen matrix, and mineralized calcium. We thus demonstrated that this device offers an adequate soil to seed primary breast cancer bone metastatic cells. In particular, patient-derived xenografts being considered as a better approach than cell lines to achieve clinically relevant results, we demonstrate the ability of this biomimetic bone niche model to host patient-derived xenografted metastatic breast cancer cells. These patient-derived xenograft cells show a long-term survival in the bone model and maintain their cycling propensity, and exhibit the same modulated drug response as in vivo. This experimental system enables access to the idiosyncratic features of the bone microenvironment and cancer bone metastasis, which has implications for drug testing.

In this study, for the first time we report on a comprehensive overview of different strategies to hyphenate droplet-based sample handling and preparation with electrophoretic separation in different formats (i.e. microchip and capillary electrophoresis). Droplet-interfaced electrophoresis is an emerging technique in which micro/nanometric droplets are used as a bridge and carrier of target analytes between sample treatment and electrokinetic separation steps, thus being expected to overcome the challenges of working dimension mismatch and low degree of module integration. This review covers all works on this topic from 2006 (the year of the first communication) up to 2020, with focus being given to three principal interfacing strategies, including droplets in immiscible phases, digital microfluidics with electrowetting-on-dielectric principle and inkjet droplet generation. Different instrumental developments for such purpose, the viewpoints on pros and cons of these designs as well as application demonstrations of droplet-interfaced electrokinetic strategies are discussed.

Simple Summary Engineered proteins possess a great therapeutic potential, but the development of such therapies is impeded during preclinical studies by the lack of in vitro models that accurately simulate the human physiology. Animal models, on the other hand, also have difficulties predicting human responses, and are ethically concerning. In this study, we employed a hybrid approach where we combined mathematical modeling with 3D in vitro models that mimic aspects of the tumor microenvironment, in order to simulate the delivery of therapeutic proteins targeting cancer cells and to predict the biological activity. By cross-comparing simulated and experimental data from 3D models, we were able to correctly predict the best dose needed to deliver toxic proteins specifically to tumor cells, while leaving the surrounding non-tumor cells untouched. This study shows the potential of combining computational approaches with novel in vitro models to advance the development of protein therapeutics. Abstract To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin repeat proteins (DARPins), in biomimetic tumor microenvironments containing fibroblasts and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) or human epithelial growth factor receptor (HER2). In multicellular tumor spheroids, we observed strong binding-site barriers in combination with low apparent diffusion coefficients of 1 µm2·s−1 and 2 µm2 ·s−1 for EpCAM- and HER2-binding DARPin, respectively. Contrasting this, in a tumor-on-a-chip model for investigating delivery in real-time, transport was characterized by hindered diffusion as a consequence of the lower local tumor cell density. Finally, simulations of the diffusion of an EpCAM-targeting DARPin fused to a fragment of Pseudomonas aeruginosa exotoxin A, which specifically kills tumor cells while leaving fibroblasts untouched, correctly predicted the need for concentrations of 10 nM or higher for extensive tumor cell killing on-chip, whereas in 2D models picomolar concentrations were sufficient. These results illustrate the power of combining in vitro models with mathematical modeling to study and predict the protein activity in complex 3D models.

Currently, fluidic control in microdevices is mainly achieved either by external pumps and valves, which are expensive and bulky, or by valves integrated in the chip. Numerous types of internal valves or actuation methods have been proposed, but they generally impose difficult compromises between performance and fabrication complexity. We propose here a new paradigm for actuation in microfluidic devices based on rigid or semi-rigid walls with transversal dimensions of hundreds of micrometres that are able to slide within a microfluidic chip and to intersect microchannels with hand-driven or translation stage-based actuation. With this new concept for reconfigurable microfluidics, the implementation of a wide range of functionalities was facilitated and allowed for no or limited dead volume, low cost and low footprint. We demonstrate here several fluidic operations, including on/off or switch valving, where channels are blocked or reconfigured depending on the sliding wall geometry. The valves sustain pressures up to 30 kPa. Pumping and reversible compartmentalisation of large microfluidic chambers were also demonstrated. This last possibility was applied to a “4D” migration assay of dendritic cells in a collagen gel. Finally, sliding walls containing a hydrogel-based membrane were developed and used to concentrate, purify and transport biomolecules from one channel to another, such functionality involving complex fluidic transport patterns not possible in earlier microfluidic devices. Overall, this toolbox is compatible with “soft lithography” technology, allowing easy implementation within usual fabrication workflows for polydimethylsiloxane chips. This new technology opens the route to a variety of microfluidic applications, with a focus on simple, hand-driven devices for point-of-care or biological laboratories with low or limited equipment and resources. A new technology, termed “sliding walls,” has been developed for operating microfluidic devices (which function at sub-millimetre scales), whereby rigid or semi-rigid walls, hundreds of micrometres thick, slide within a microfluidic chip. Fluid control in micro-devices is currently achieved largely by expensive, bulky external valves and pumps or by valves integrated in the chip. Such internal valves or actuation methods generally have shortcomings related to performance and complex fabrication. A team headed by Stephanie Descroix and Jean-Louis Viovy at the Institut Curie—Institut Pierre Gilles de Gennes, Paris applied its sliding wall technique and was able to execute several fluidic operations in which channels could be blocked or reconfigured through the sliding wall geometry. The authors believe that their technology has the potential for various microfluidic operations in biological laboratories with limited equipment and resources.

International audience; We present an innovative droplet microfluidic device based on integrated soft-magnetic structures allowing extraction, redispersion and depletion processes of magnetic particles. Following a numerically-assisted conception, the device was at first fabricated and experimentally characterized, showing reliable and efficient magnetic particles extraction with a low carryover of the initial matrix (

Although fibroblast heterogeneity is recognized in primary tumors, both its characterization in and its impact on metastases remain unknown. Here, combining flow cytometry, immunohistochemistry and RNA-sequencing on breast cancer samples, we identify four Cancer-Associated Fibroblast (CAF) subpopulations in metastatic lymph nodes (LN). Two myofibroblastic subsets, CAF-S1 and CAF-S4, accumulate in LN and correlate with cancer cell invasion. By developing functional assays on primary cultures, we demonstrate that these subsets promote metastasis through distinct functions. While CAF-S1 stimulate cancer cell migration and initiate an epithelial-to-mesenchymal transition through CXCL12 and TGFβ pathways, highly contractile CAF-S4 induce cancer cell invasion in 3-dimensions via NOTCH signaling. Patients with high levels of CAFs, particularly CAF-S4, in LN at diagnosis are prone to develop late distant metastases. Our findings suggest that CAF subset accumulation in LN is a prognostic marker, suggesting that CAF subsets could be examined in axillary LN at diagnosis., Cancer associated fibroblasts are known to promote the progression of cancer. Here, the authors show that two particular subsets of cancer associated fibroblasts induce metastasis but work via distinct mechanisms including, chemokine signalling and Notch signalling.

International audience

An original method of monolith impregnation in microsystem for the analysis of radionuclides in nitric acid is reported. Three microcolumns made of monolith poly(AMA-co-EDMA) were impregnated in COC microsystems. The robustness of the microsystems in nitric acid media until 8 M was demonstrated. High exchange capacity and affinity for tetravalent and hexavalent actinides in concentrated nitric media were obtained. The retention characteristics of the microcolumns impregnated by TBP, TBP-CMPO and DAAP were compared with those of the equivalent commercial particulate resins TBP™, TRU™ and UTEVA™ respectively. The separation of U, Th and Eu was validated in a classical microsystem and a procedure is proposed in a centrifugal microsystem.

An experimental design is described for optimization of the microscopic morphology of a methacrylate monolith that was elaborated for chromatographic separation of radionuclides in nitric acid media. This paper presents a systematic study of the synthesis of the polymeric porous monolith poly(ethylene glycol dimethacrylate-co-allyl methacrylate) used as solid-phase support and a post-functionalization of the monolith in microsystem with tributyl phosphate extractant. Polymerization time and chemical composition of the polymerizable mixture that comprises water, 1,4-butanediol, 1-propanol, monomers were chosen as the most relevant experimental factors of the photochemical process. Using the globules area as a significant response of an experimental design, the monolith morphology can be predicted. A new versatile and robust impregnation process was developed in microsystem. The designed micro chromatographic system showed a good resistance in concentrated nitric acid and a great loading capacity compared to commercially available solution (150 mg U versus 75 mgU/g resin).

In this study, single-step magnetic-beads based immunoassays was developed and for the first time adapted to microfluidic droplet operation for sequential determination of well-established amyloid-β peptide biomarkers ( i.e . monomeric Aβ 1–42 and Aβ 1–40) for molecular diagnosis of Alzheimer’s disease (AD). With the developed sandwich assay protocol, the capture antibodies ( i.e. monoclonal anti-Aβ antibodies that are specific for N-terminus of Aβ 1–42 and Aβ 1–40) grafted onto magnetic beads and the detection antibodies ( i.e. beta amyloid 1–16 monoclonal antibody labeled with horseradish peroxidase) can simultaneously bind to monomeric β peptides in a single step. Aβ 1–42 and Aβ 1–40 in cerebrospinal fluid (CSF) samples were successfully detected using the developed batchwise immunoassay approach. With the aim at expanding the spectrum of traced Aβ peptides beyond Aβ 1–42 and Aβ 1–40 for more precise diagnostics of AD, the developed sandwich assay was for the first time coupled as a downstream module to peptide fractionation and collection using capillary isoelectric focusing. Demonstration of this methodological combination was carried out with Aβ 1–40, Aβ 1–40 and Aβ 5–40. The immunoassay in batchwise format was downscaled into a purpose-made microfluidic droplet platform allowing significant sample volume reduction and higher throughput. Using a series of 4 programmable magnetic tweezers to manipulate a train of nano-scale confined droplets containing magnetic beads, sample, washing and detection solutions, a sequence of 8 analyses could be realized within 45 min. This droplet based immunoassay was realized in a dedicated platform integrating an in-house-made light emitting diode (LED)-based fluorescent detector, replacing conventional microscopic setup so as to significantly reduce the construction cost and simplify the detection protocol. Using this microfluidic configuration coupled with LED-based detection, information on both Aβ 1–42 and Aβ 1–40 concentrations can be collected in a single sequence with less than 1 μL of sample.

The sealing of microfluidic devices remains a complex and time-consuming process requiring specific equipment and protocols: a universal method is thus highly desirable. We propose here the use of a commercially available sealing tape as a robust, versatile, reversible solution, compatible with cell and molecular biology protocols, and requiring only the application of manually achievable pressures. The performance of the seal was tested with regards to the most commonly used chip materials. For most materials, the bonding resisted 5 bars at room temperature and 1 bar at 95 °C. This method should find numerous uses, ranging from fast prototyping in the laboratory to implementation in low technology environments or industrial production.

International audience; Fluidization, a process in which a granular solid phase behaves like a fluid under the influence of an imposed upward fluid flow, is routinely used in many chemical and biological engineering applications. It brings, to applications involving fluid–solid exchanges, advantages such as high surface to volume ratio, constant mixing, low flow resistance, continuous operation and high heat transfer. We present here the physics of a new miniaturized, microfluidic fluidized bed, in which gravity is replaced by a magnetic field created by an external permanent magnet, and the solid phase is composed of magnetic microbeads with diameters ranging from 1 to 5 μm. These beads can be functionalized with different ligands, catalysts or enzymes, in order to use the fluidized bed as a continuous purification column or bioreactor. It allows flow-through operations at flow rates ranging from 100 nL min −1 up to 5 μL min −1 at low driving pressures (

International audience; We report measurements of the linear viscoelastic modulus of collagen gels. Collagen gels are cross-linked networks of bundles formed from the association of single filaments. Their structure is heterogeneous at microscopic length scales, and measurements of their viscoelasticity at a larger scale are required. We use a technique that we recently developed based on the measurement of surface thermal fluctuations and called surface fluctuation specular reflection [Pottier et al., Soft Matter 7, 7843–7850 (2011)] to characterize the linear viscoelastic properties of collagen gels for frequencies ranging from 1 Hz to 30 kHz. These properties are determined at a mesoscopic scale (∼100 μm) at which the gels are homogeneous. We further study the influence of both collagen origin and concentration in the light of previous findings on these systems.

International audience; Preeclampsia is a hypertensive pregnancy disease associated with a massive increase in sFlt-1 (soluble form of the vascular endothelial growth factor 1) in the maternal circulation, responsible for angiogenic imbalance and endothelial dysfunction. Pilot studies suggest that extracorporeal apheresis may reduce circulating sFlt-1 and prolong pregnancy. Nonspecific apheresis systems have potential adverse effects because of the capture of many other molecules. Our concept is based on a specific and competitive apheresis approach using VEGF (vascular endothelial growth factor) functionalized magnetic beads to capture sFlt-1 while releasing endogenous PlGF (placental growth factor) to restore a physiological angiogenic balance. Magnetic beads were functionalized with VEGF to capture sFlt-1. Experiments were performed using PBS, conditioned media from human trophoblastic cells, and human plasma. The proof of concept was validated in dynamic conditions in a microfluidic device as an approach mimicking real apheresis. Magnetic beads were functionalized with VEGF and characterized to evaluate their surface ligand density and recognition capabilities. VEGF-coated magnetic beads proved to be an efficient support in capturing sFlt-1 and releasing PlGF. In static conditions, sFlt-1 concentration decreased by 33±13%, whereas PlGF concentration increased by 27±10%. In dynamic conditions, the performances were improved, with 40% reduction of sFlt-1 and up to 2-fold increase of free PlGF. The sFlt-1/PlGF ratio was reduced by 63% in the plasma of preeclamptic patients. Apheresis was also associated with VEGF release. A ligand-based approach using VEGF-coated beads is an effective approach to the capture of sFlt-1 and the release of endogenous PlGF. It offers new perspectives for the treatment of preeclampsia.

This study reports on the conception of magneto-Capillary Electrophoresis (magneto-CE), an approach integrating immuno-capture on circulating bio-functionalized magnetic beads into a unique capillary for preconcentration and electrokinetic separation. This hybrid mode is an evolution of in-capillary magnetic bead-based operation from static cluster format to dynamic configuration where beads are allowed to controllably circulate inside a CE capillary for interaction improvement. To implement the magneto-CE operation, a purpose-made instrument was constructed, allowing visual observation of the movement of the magnetic beads. We applied a new methodological strategy for determination of the amyloid beta peptide (A beta 1-42), which is as an established biomarker for molecular diagnosis of Alzheimer's disease (AD). The methodology is based on magneto-immuno-capture of fluorescently labeled A beta 1-42 followed by a chemical elution with a basic solution prior to CE separation with laser induced fluorescent (LIF) detection. The superiority of this dynamic configuration of magneto-CE was demonstrated for this target analyte, with sample pretreatment and separation being performed in-capillary without any delay in between and without any waste of pretreated sample, which otherwise would not be the case with offline/batch-wise operation. (C) 2019 Elsevier B.V. All rights reserved.

International audience; Droplet microfluidics is a powerful technology that finds many applications in chemistry and biomedicine. Among different configurations, droplets confined in a capillary (or plugs) present a number of advantages: they allow positional identification and simplify the integration of complex multi-steps protocols. However, these protocols rely on the control of droplet speed, which is affected by a complex and still debated interplay of various physico-chemical parameters like droplet length, viscosity ratio between droplets and carrier fluid, flow rate and interfacial tension. We present here a systematic investigation of the droplet speed as a function of their length and interfacial tension, and propose a novel, simple and robust methodology to control the relative distance between consecutive droplets flowing in microfluidic channels through the addition of surfactants either into the dispersed and/or into the continuous phases. As a proof of concept application, we present the possibility to accurately trigger in space and time the merging of two confined droplets flowing in a uniform cross-section circular capillary. This approach is further validated by monitoring a conventional enzymatic reaction used to quantify the concentration of H 2 O 2 in a biological sample, showing its potentialities in both continuous and stopped assay methods.

An efficient and reproducible photopolymerized poly(ethylene glycol methacrylate methacrylate-co- allyl methacrylate) monolith was synthesized and a photografting process based on the ene-thiol click-chemistry has been performed to give anion exchange properties to the monolith. Since their introduction in the early 1990s polymethacrylate monoliths have emerged as a powerful alternative for microscale separations or sample treatment. Their relatively simple implementation in columns with small internal diameters makes them particularly attractive for the new chromatographic challenges of complex matrices analysis and on-chip separations. Despite their relatively poor ion-exchange capacity due to their highly porous structure, their use as anion exchangers is of large interest for nuclear analysis as numerous separations are based on this process. This paper presents a systematic study of the synthesis of the polymeric porous monolith and the versatile and robust functionalization method developed for the specific strong acidic media used in radiochemical procedures. The robustness of the stationary phase was tested in concentrated nitric acid.

International audience; Tumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent me-tastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity.

Droplet microfluidics allows the implementation of several biochemical and bioanalytical protocols in microfluidic devices. However, this integration typically requires the use of microfluidic valves, which allow the control of different operations on the same channels network. Unfortunately, the valves commercially available are almost exclusively suitable for-single phase flow, and only one valve system compatible with multi-phase systems has been presented so far in the literature. We present here a new pinch-like valve that withstands high pressures (tested up to 4 bar), allows droplet manipulation and combines the advantages of conventional pinch valves regarding the absence of dead volume, while making negligible the backflow generated (less than 2 nL). The conception and design of the presented valve, based on numerical simulations, is performed by an optimized microfabrication strategy, combining PDMS soft-lithography and 3D printing technologies. The valves had been tested regarding their mechanical stability and proving their capability of allowing droplets passing through them, without any perturbation. Finally, the very small back-flow volume generated during the actuation, has been exploited for various applications in droplets microfluidics, like droplet handling, droplet-on-demand generation and sorting.

International audience; The success of microfluidic immunocapture based on magnetic beads depends primarily on a sophisticated microscale separation system and on the quality of the magnetic immunosorbent. A microfluidic chip containing a magnetically stabilized fluidized bed (μMSFB), developed for the capture and on-chip amplification of bacteria, was recently described by Pereiro et al.. The present work shows the thorough development of anti-Salmonella magnetic immunosorbents with the optimal capture efficiency and selectivity. Based on the corresponding ISO standards, these parameters have to be high enough to capture even a few cells of bacteria in a proper aliquot of sample, e.g. milk. The selection of specific anti-Salmonella IgG molecules and the conditions for covalent bonding were the key steps in preparing an immunosorbent of the desired quality. The protocol for immunocapturing was first thoroughly optimized and studied in a batchwise arrangement, and then the carrier was integrated into the μMSFB chip. The combination of the unique design of the chip (guaranteeing the collision of cells with magnetic beads) with the advanced immunosorbent led to a Salmonella cell capture efficiency of up to 99%. These high values were achieved repeatedly even in samples of milk differing in fat content. The rate of nonspecific capture of Escherichia coli (i.e. the negative control) was only 2%.

A major challenge in cancer research is the complexity of the tumor microenvironment and the necessity to take into account the host immunological setting. An innovative way to address these problems is to exploit the emerging technology of organ-on-chip to achieve co-cultures in microfluidic devises that recapitulate ex vivo the tumor ecosystem. We generated tumors-on-chip integrating four cell populations (cancer, immune, endothelial cells and fibroblasts) in a 3D collagen matrix, reconstituting HER2 breast cancer ecosystem. By time-lapse microscopy, differential live staining and a novel automated analysis method, we visualized, and quantified the complex dynamics of this ecosystem, in absence or in presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (≳ 60 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) on the contrary inhibited cancer-immune interactions, antagonizing the action of the trastuzumab. These observations constitute a proof-of-concept that tumors-on-chip are novel powerful platforms that can be exploited to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level responses to anti-cancer drugs, and to dissect the roles of the various cellular components of the stroma.

International audience; A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolithbased chip. Their performances were first demonstrated with N-benzoyl-DL-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.

International audience; Fluorescence measurement is the main technology for post-amplification DNA detection in automated systems. Direct electrical reading of DNA concentration in solution could be an interesting alternative to go toward more miniaturized or less expensive devices, in particular in the pathogen detection field. Here we present the detection of short bacterial biomarkers with a direct impedancemetric measurement, within solutions of amplified and elongated DNA sequences in a microchannel. This technology relies on the electrohydrodynamic instability occurring in solutions of long charged macromolecules in a strong electric field. This instability specifically induces the aggregation of long DNAs and triggers conductivity variations that can be monitored by on-contact conductometry. An innovative isothermal amplification and elongation strategy was developed, combining SDA and HRCA reactions, in order to yield long DNAs suitable to be detected by the above principle, from a dilute initial DNA target. In contrast with previous label-free detection methods, this new strategy is very robust to matrix effects, thanks to the unique molecular weight dependence of the instability, coupled with this specific DNA amplification strategy. We demonstrate the detection of a 1 pM gene sequence specific to Staphylococcus aureus, in a portable system.